This study investigated the influence of sodium bicarbonate (NaHCO3) and progesterone on acrosome reaction and proportion of polyunsaturated fatty acid (PUFA) composition boar sperm. The sperm were diluted with semen extender and incubated with NaHCO3 and progesterone at 38℃, 5% CO2 for 6 h. Plasma membrane integrity and acrosome reaction were analyzed using SYBR14/propidium iodide (PI) and FITC-PNA/PI doubling staining method, and proportion of PUFA was analyzed using gas chromatography. In results, Plasma membrane integrity was significantly decreased in 50 mM NaHCO3 group and acrosome reaction was significantly increased by over the 100 mM NaHCO3 group compared to control group (p < 0.05). In addition, progesterone significantly increased decreased plasma membrane integrity at 100 mM progesterone and acrosome reaction at over the 5.0 µM progesterone (p < 0.05), but there was no difference among the 5.0 to 100 µM groups. PUFAs were significantly decreased in 100 mM NaHCO3 and 50 µM progesterone treatments compared to control group. In summary NaHCO3 and progesterone induce acrosome reaction and reduce PUFA composition in boar sperm, therefore, the results maybe help to understand basically knowledge for the acrosome reaction and PUFA composition in boar sperm.

Cryopreservation is mainly used for preservation of boar sperm. However, this method stresses the sperm by reactive oxygen species (ROS), and the conception rate and the litter size are not more efficient than the liquid preservation of spermatozoa. Therefore, we use chitosan which is a natural product derived antioxidant compound. We used GnHA (chitosan+hyaluronic acid) and GnHG (chitosan hydrogel) as chitosan complexes to cryopreserve boar sperm for improve sperm metabolism and function. Sperm parameter (sperm motility, progressive motility, path velocity, straight-line velocity, curvilinear velocity) is measured by computer-assisted sperm analysis (CASA) using frozen sperm with GnHA or GnHG (0, 0.25, 0.5, 1 mg/mL), respectively. Also, lipid peroxidation analysis using malondialdehyde (MDA) is performed to confirm the antioxidative effect of chitosan in frozen spermatozoa. CASA analysis showed GnHA and GnHG are effective against cryopreserved boar sperm. And antioxidant effect is measured by lipid peroxidation analysis. GnHA and GnHG, which is chitosan complex are effective for boar sperm cryopreservation by antioxidant effect.

Estrogen receptor 2 (ESR2) is involved in oestrogen-related apoptosis in cell cycle spermatogenesis but their effects have not yet confirmed in pig. Therefore, this study was performed to investigatetheir association with semen motility and kinematic characteristics. DNA samples from 126 Duroc pigs with records of sperm motility and kinematic characteristics [Total motile spermatozoa (MOT), Curvilinear velocity(VCL), Straight-line velocity(VSL), the ratio between VSL and VCL(LIN), Amplitude of Lateral Head displacement(ALH)] were used in present study. A single nucleotide polymorphism (g.35547A>G) was associated with MOT, VCL, VAP and ALH in Duroc population (p < 0.05). Therefore, we suggest that the porcine ESR2 may be used as a molecular marker for Duroc boar semen quality, although its functional effect was not clear yet. These results will improve the understanding of the functions of the ESR2 in spermatogenesis within the reproductive tracts, and will shed light on ESR2 as a candidate gene in the selection of good sperm quality boars.

During the freezing and thawing process, fatty acids in the plasma membrane of sperm are released, which results in a functional damage of sperm. Sperm with functional loss due to cryo-damage result in a decrease in fertility. Previous studies have shown that the addition of one of the fatty acid alpha-linolenic (ALA) with carrier proteins improves the stability of plasma membrane and reduces the damage. In this experiment, we focused on the functional aspects of the plasma membrane of sperm and experimented with motility and morphology. For preparation of ALA-carrier protein complex, 3 ng/ml ALA was mixed with 0.7 μg/ml bovine serum albumin (BSA) or 14 ng/ml methyl-β-cyclodextrin (MBCD) in distilled water. The boar semen was purchased from GUMBO Company. Boar semen was cryo-preserved in 20% egg yolk freezing extender containing ALA, BSA, MBCD, ALA+BSA, ALA+MBCD. The frozen boar sperm was thawed at 37.5 ℃ for 45 sec in water-bath. The sperm motility and morphological abnormalities were evaluated under a phase-contrast microscope at 200 × magnification and randomly counts of 200 sperm each sample. In results, motility of frozen-thawed sperm was increased in all treatment groups. In particular, there has been significant improvement in ALA+BSA and ALA+MBCD treatment groups than control (p<0.05). However, there was no significant difference in ALA, BSA and MBCD treatment groups. Morphological normalities in frozen-thawed sperm was reduced in complex treatment groups (p<0.05). However, there was no significant difference in single treatment groups. In both motility and morphology characteristics, ALA+BSA and ALA+MBCD treatment group was higher than all treatment groups. In conclusion, the addition of ALA with carrier proteins during cryopreservation has a positive effect in its functional aspect. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Education) (2016R1D1A1B03931746).

Cryopreservation is mainly used for preservation of boar sperm. However, this method stresses the sperm by reactive oxygen species (ROS), and the conception rate and the litter size are not more efficient than the liquid preservation of spermatozoa. Therefore, we use chitosan which is a natural product derived antioxidant compound. We used GnHA and GnHG as chitosan complexes to cryopreserve boar sperm for improve sperm metabolism and function. Sperm parameter (sperm motility, progressive motility, path velocity, straight-line velocity, curvilinear velocity) is measured by computer-assisted sperm analysis (CASA) using frozen sperm with GnHA or GnHG (0, 0.25, 0.5, 1 mg/mL). Also, lipid peroxidation analysis using malondialdehyde (MDA) is performed to confirm the antioxidative effect of chitosan in frozen spermatozoa. Sperm motility was higher in GnHA 0.25 mg/mL and GnHG 0.5 mg/mL compared to control. In addition, GnHG 0.5 mg/mL was significantly decreased in lipid peroxidation analysis. The results suggest that GnHA and GnHG are effective for boar sperm cryopreservation by antioxidant effect.

The aim of this study was performed to evaluate the effects of ice-binding protein from the arctic yeast Leucosporidium (LeIBP) supplementation on cryopreservation of boar sperm. The collected semen was diluted (1.5×108/ml) in lactose egg yolk (LEY) and cooled at 5°C for 3 h. The cooled semen was then diluted (1×108/ml) in LeIBP containing LEY with 9% glycerol and maintained at 5°C for 30 min. The semen was divided into six experimental groups (control, 0.001, 0.005, 0.01, 0.05 and 0.1 mg/ml of LeIBP). The straws were kept on above the liquid nitrogen (LN2) vapors for 20 minutes and then plunged into LN2. After thawing, computer-assisted sperm analysis was used for sperm motility and flow cytometry was performed to assess the viability, acrosome integrity (FITC-PSA/PI), ROS (DCF/PI), lipid peroxidation (BODIPY C11/PI) and apoptosis (Annexin V/PI), respectively. No significant responses were observed for sperm motility. However, sperm viability was significantly increased on 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). In addition, acrosome integrity was significantly increases LeIBP groups (P < 0.05) and both ROS and lipid peroxidation level were lower in all LeIBP groups than those of control (P < 0.05). On the other hand, a significant higher apoptosis rate was observed in 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). It can be assumed that a supplementation of LeIBP in boar sperm freezing extender is an effective method to increase the sperm qualities after cryopreservation.

This study was to investigate effect of tunicamycin (TM) on sperm viability, mitochondrial activity and motility in boar semen. Collected sperm were incubated with semen extender containing 0, 1, 2, and 5 μM TM for 3, 6 and 9 h. Sperm viability was analyzed using SYBR14/PI doubling staining, and mitochondrial activity was detected using Rhodamine123/PI staining methods. Sperm viability, mitochondrial activity and motility were measured every 3 h during incubation. In results, boar sperm viability, mitochondrial activity and motility were significantly decreased in 2 and 5 μM TM groups compare to control group at all incubation time (p<0.05). In addition, mitochondrial activity and motility were significantly decreased in 1, 2, and 5 μM TM groups compare to control group at 9 h after incubation (p<0.05). These results suggest that TM can inhibit sperm viability, mitochondrial activity and motility in boar semen, and it may influence on the fertility of sperm.

The objective of this study was to investigate the influence of the addition of caffeine (alkaloid family) to the ejaculates of boar sperm quality as well as investigate their optimum concentrations for increasing the movement of sperms. Semen was collected from 9 boars by the gloved-hand technique one week interval. Semen followed by cryopreservation with egg yolk extender freezing medium using freezing protocol. The collected semen were frozen on the same day. Motility was assessed for % motile cell characteristics using computer-assisted semen analysis (CASA; SAIS SI-100, Medical supply, Korea). Frozen boar sperms were thawed in Beltsville Thawing Solution (BTS) with 5, 10, and 15mM caffeine were then incubated at 38 celsius degree for 20 minutes. In experiment 1, semen were diluted BTS and addition of different concentration of caffeine to the pre-freeze semen cryopreservation. In experiment 2, incubation of frozen-thawed sperm in BTS supplemented with different concentration of caffeine for 20 minutes improved (P<0.05) after semen cryopreservation-thawing on sperm quality. After thawing significantly increased progressive and total motility. The addition of 10 mM caffeine to cryopreserved semen after thawing significantly increased progressive and total motility compared with other treatment. These result suggest that caffeine enhanced post-thaw motility of cryopreserved boar sperm when added after thawing.

This study was conducted to evaluate effect of α-linolenic acid (ALA) and bovine serum albumin (BSA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved using freezing extender containing 3 ng/mL ALA and/or 20 μg/mL BSA. Cryopreserved boar sperms were thawed in 37°C water-bath for 45 sec to analysis. Viability, acrosome reaction, and mitochondrial intact were analyzed using flow cytometry. In results, viability of frozen-thawed boar sperm was significantly higher in only ALA+BSA supplement group than control group (p<0.05), whereas there was no difference either in ALA or BSA supplement. However, acrosome reacted sperm in both of live and all sperm population were significantly decreased in all treatment groups than control (p<0.05). Interestingly, mitochondrial intact of boar sperm was enhanced in ALA and ALA+BSA groups compared with control (p<0.05). In this study, we showed that supplementation of ALA and BSA in freezing extender enhanced the sperm viability, mitochondrial intact and decrease acrosomal membrane damage. In conclusion, our findings suggest that quality of frozen-thawed sperm in mammalians could improve by using of ALA and BSA.

For evaluating the boar semen quality, sperm motility (MOT) is an important parameter because the movement of spermatozoa indicates active metabolism, membrane integrity and fertilizing capacity. Phospholipase C zeta (PLCz) is important enzyme in spermatogenesis, but their effect has not been confirmed in pigs yet. Therefore, this study was aimed to analyze their association with sperm motility and kinematic characteristics. DNA samples from 124 Duroc pigs with records of sperm motility and kinematic characteristics [Total motile spermatozoa (MOT), Curvilinear velocity (VCL), Straight-line velocity (VSL), the ratio between VSL and VCL (LIN), Amplitude of Lateral Head displacement (ALH)] were subjected. A SNP in non-coding region of PLCz g.158 A > C was associated with MOT (p < 0.05), VCL (p < 0.01), LIN (p < 0.01) and ALH (p < 0.05) in Duroc population. Therefore, we suggest that the intron region of the porcine PLCz gene may be used as a molecular marker for Duroc boar semen quality, although its functional effect was not defined yet. Whether the association is due to the candidate gene or not require further verification. Thus, it will be of interest to continue association studies in the regions surrounding those genes.

The purpose of this study was to examine the effects of taurine and vitamin E on sperm characteristics damaged by bromopropane (BP) in pig. We evaluated toxicity of BP on viability, membrane integrity and mitochondrial activity of spermatozoa. 1-BP (0, 2.5, 5.0, 10, and 50 μM), 2-BP (0, 2.5, 5.0, 10, and 50 μM), taurine (0, 5.0, 10, and 25 μM) and vitamin E (0, 50, 100, and 200 μM) were treated in fresh boar semen for 6 h. 10 and 50 μM of 1-BP and 2-BP inhibited sperm viability, membrane integrity and mitochondrial activity in fresh boar semen (P<0.05). 25 μM of taurine increased sperm viability and membrane integrity (P<0.05), 100 μM of vitamin E enhanced viability and mitochondrial activity of sperm (P<0.05). Finally, 10 μM of 1-BP and 2-BP was co-treated with taurine (25 μM) and vitamin E (100 μM) in the fresh boar semen. The co-treated samples did affected viability, membrane integrity and mitochondrial activity of sperm. In conclusion, taurine and vitamin E can improve and maintain sperm quality in fresh boar semen.

The objective of this study was to evaluate the efficiency of sperm cryosurvival in boar sperm separated by Percoll containing antioxidant enzymes. The boar semen was collected into a pre-warmed (37℃) thermos bottle by gloved-hand method and was separated by 65% Percoll with superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) before freezing. The frozen sperm was thawed at 38.5℃ for 45 sec in water-bath for sperm characteristic analysis. The sperm were estimated with SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction, Rhodamine123/PI double staining for mitochondrial integrity and were analyzed using flow cytometry. In results, sperm viability, acrosome reaction and mitochondrial integrity were improved in separated sperm groups compared with unseparated sperm by Percoll (UP) group. Especially, viability was significantly higher in sperm separated by Percoll containing 400 IU CAT group compared with other groups (P<0.05). And acrosome reaction was decreased in sperm separated by Percoll with 300 IU SOD, 400 IU CAT and 0.5 mM GSH groups compared with other groups, however, there were no significantly difference mitochondrial integrity among sperm separated by Percoll with antioxidant enzymes. In conclusion, we suggest that use of Percoll containing antioxidant enzymes for sperm separation will be beneficial for sperm cryopreservation in pigs.

The objective of this study was to investigate the efficiency of nicotinic acid on sperm cryosurvival and fertilization ability in frozen-thawed boar semen. Boar semen was collected by glove-hand method and was frozen using freezing solution treated to 0, 5, 10 and 20 mM of nicotinic acid. The frozen sperm for sperm characteristic analysis was thawed such as viability, acrosome reaction, and mitochondrial integrity. The frozen-thawed sperm was estimated by SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction and Rhodamine123/PI double staining for mitochondrial integrity using a flow cytometry. The embryo was estimated in vitro development and DCFDA staining for reactive oxygen species assessment. As results, frozen-thawed sperm viability was significantly higher in 5 and 10 mM (61.1 ± 1.5%, 64.7 ± 2.0%) of nicotinic acid than other groups (0 mM, 52.1 ± 2.3%; 20 mM, 47.8 ± 5.1%, P<0.05). The live sperm with acrosome reaction was significantly higher in 5 and 10 mM of nicotinic acid (26.1 ± 1.8%, 24.9 ± 1.5%) than other groups (0 mM, 35.3 ± 0.8%; 20 mM, 36.5 ± 1.9%, P<0.05). The live sperm with mitochondrial integrity was significantly higher in 5 and 10 mM (84.2 ± 3.6%, 88.4 ± 2.3%) of nicotinic acid than other groups (0 mM, 77.3 ± 4.4%; 20 mM, 73.3 ± 3.6%, P<0.05). Blastocyst rate of in vitro development was significantly higher in 10 mM (17.0 ± 1.3%) of nicotinic acid than other groups (0 mM, 9.4 ± 0.5%; 5mM, 12.6 ± 0.8%; 20 mM, 5.0 ± 1.0%, P<0.05). Moreover, total cell number was higher in 5 and 10 mM (53.6 ± 2.9%, 57.9 ± 2.8%) of nicotinic acid than other groups (0 mM, 41.0 ± 1.4%; 20 mM, 23.2 ± 2.8%, P<0.05). Hydrogen peroxide in embryos was lower in 5 mM nicotinic acid (0.7 ± 0.1%) than other groups (0 mM, 1.0 ± 0.1%; 10mM, 0.9 ± 0.0%; 20 mM, 1.4 ± 1.0%, P<0.05). In conclusion, nicotinic acid-treated semen improves cryosurvival and quality of spermatozoa. Also, the fertilized oocytes with nicotinic acid improve quality of embryo and blastocyst formation.

The boar sperm has more lipid droplets and specialty of seminal plasma compared with other species, causing difficulties of freezing sperm and decreases for the utilization of frozen semen into the artificial insemination. However, several studies reported significant results for the recovery of sperm motility and reproductive by addition of cryoprotectants and seminal plasma after thawing. This study was designed to investigate the effects of supplementation of trehalose or glycerol in the LEY (lactose and egg yolk in BTS) solution for the conventional freezing and vitrification process. Two boars aged 16 months were used to collect semen for 2 times in a week. The samples were allotted to 3 freezing solutions (LEY + glycerol 10.5% + OEP 1.5%, LEY + trehalose 1M + OEP 1.5%, and sucrose 1.5M + trehalose 1 M + OEP 1.5%) after centrifugation at 800 g for 10 minutes. Semen was equilibrated in freezing solutions for 10 minutes and injected into plastic straws with 2∼3 air bubbles to minimize freezing damages. Vitrification was performed to locate sperm in 5 cm above LN2 for 5 minutes, and the conventional freezing was conducted with an automatic freezer. Motility and survival rates were measured by CASA (Computer assisted sperm an alyzing system) and FITC (Fluorescein isothiocyanate), respectively after thawing semen at 50℃ for 12 seconds. The results were analyzed by ANOVA with STATVIEW statistical program. The vitrificatioin solution (LEY + 10.5% glycerol + 1.5% OEP) presented higher motility (20.9%) than other solutions while the solution (LEY + 1M trehalose + 1.5% OEP) showed the lowest (motility : 5.2%). However, survival rates of vitrified sperms detected by FITC showed 1~4% live sperms in almost of dead sperms at all vitrification solutions’ groups, but survival rate of freezing solution of LEY + 1M trehalose + 1.5% OEP LEY and LEY + 10.5% glycerol + 1.5% OEP were showed 49%, and 79%, respectively. There were differences (P<0.05) survival rate of conventional freezing in LEY + 10.5% glycerol + 1.5% OEP and LEY + 1M trehalose + 1.5% OEP and the remaining showed no differences. The results suggested that vitrified boar semen was not enough to be utilized for the artificial insemination, but it showed possibility to utilize for ICSI and conventional freezing with glycerol would be useful method for artificial insemination in pig while we choose the outstanding semen against tolerance to freezing damages.

The objective of this study was to determine the effects of E. coli on boar sperm quality and reproductive performance in sows after artificial insemination. Three different levels of E. coli were artificially inoculated to semen with following concentrations; Control, 500, 5,000 and 50,000 colony forming unit (cfu)/ml. Semen samples were preserved at 17℃ for 5 days. Sperm motility was significantly decreased (p<0.05) on day 3 in the group inoculated with 5,000cfu/ml compared to control groups. In all treatment groups, sperm motility was gradually decreased as storage time increased, but the decline pattern was more drastic in the groups inoculated with 5,000 and 50,000 cfu/ml groups from day 3 (p<0.05) compared to control group. After 3 day of storage at 17℃, sperm viability in sample inoculated with the highest concentration (50,000 cfu/ml) of bacteria was less (p<0.05) than that of control group. The pH of semen sample pH was maintained 7.2～7.5 in all groups during the experimental period. No differences (p>0.05) were found for both storage time and bacterial concentration. The pregnancy rate and live born piglets tend to decrease by increasing the concentration of E. coli in semen. In particular, the rate of pregnancy was lower in the group inoculated with 50,000 cfu/ml (58.3%) compare to the other groups (81.8, 75.0, 76.5%). These results suggest that the contamination of E. coli in boar semen negatively affects fertilizing ability of boar sperm and the reproductive performance obtained from sows after artificial insemination.

Quercetin and genistein, plentifully present in fruits and vegetables, are flavonoid family members that have antioxidative function and plant-derived phytoestrogen activity. The antioxidative effects of quercetin and genistein on boar sperm characteristics and in vitro development of IVF embryo were investigated. The sperm motility was increased by addition of genistein 50 μM for 6 hr incubation compared to control (p<0.05). The sperm viability was increased by addition of quercetin 1 and 50 μM and genestein 1 and 50 μM for 3 hr incubation. In addition, the sperm viability seemed to be increased dose-dependantly by addition of quercetin or genistein 1 and 50 μM, respectively (p<0.05). The membrane integrities were not increased by quercetin or genistein treatments for 3 hr or 6 hr incubation period except for quercetin 1 μM for 3 hr incubation. In mitochondrial activities, addition of quercetin 50 μM for 6 hr incubation increased mitochondrial activity but decreased at 100 μM concentration compared with control (p<0.05). When porcine IVF embryos were cultured in PZM-3 medium supplemented with low concentrations of quercetin (1∼10 μM), the developmental rates to morula and blastocyst increased but significantly decreased at high concentrations of quercetin (25∼50 μM). The highest developmental rate to blastocysts among all concentrations of quercetin was shown at quercetin 10 μM (p<0.05). The developmental rates to morula or blastocysts at low (0.01∼1 μM) and high (5∼10 μM) concentrations of genistein were not significantly different among all treatment group and genistein did not affect on IVF embryo development. These results suggest that quercetin and genistein seem to have positive effects at certain concentrations on sperm characteristics such as motility, viability and mitochondrial activity. In addition, low concentrations of quercetin (1, 5 and 10 μM) in this experiment, seem to have beneficial effect on porcine IVF embryo development but genistein did not affect on it at all given concentrations (0.01∼10 μM).

The objective of this study was to investigate the relationship between fertility and protein pattern change using in vitro fertilization, analysis of sperm characteristics and two-dimensional gel electrophoresis in different pig types. In results, the viability and mitochondria integrity of sperm were higher significantly (p<0.05) but the portions of ac-rosome reaction was lower significantly (p<0.05) in Duroc and F1 (potbellied×PWG miniature pig) than PWG minia-ture. On in vitro fertilization to investigate fertility, the fertility of F1 semen war higher significantly (p<0.05) than in Duroc and PWG miniature pig. On the other hand, protein patterns showed similar function among the different boar semen. Especially, the heat shock 70 kDa 1-like and G patch domain-containing protein 4 were significantly (p<0.05) higher expressed in F1 than in Duroc and PWG miniature pig. The proteins associated with mitochondria in Duroc were significantly (p<0.05) higher expressed than in F1 and PWG miniature pig. The developmental rates to blastocyst stage of oocytes fertilized with sperm of F1 pig were significantly (p<0.05) higher than in PWG miniature pig. However, phosphoglycerate kinase 2 and zinc finger protein 431 were significantly (p<0.05) higher expressed in PWG miniature pig than in F1 and Duroc pigs. In conclusion, the results of the present study indicate that different proteins were expressed in different pig types, and were associated with a sperm functions and embryo develop-ment.

The objective of this study was to compare the effect of semen extenders on the sperm motility, viability, acrosome integrity and functional integrity of plasma membrane (HOST: hypo-osmotic swelling test) during liquid preservation of Korean Native boar semen. In this experiment, semen was diluted in Androhep plus, Beltsville Thawing Solution (BTS), ModenaTM, Seminark and Vitasem LD. Sperm-rich fractions were collected from three Korean Native boars and sub-samples were diluted (30×106 spermatozoa/ml) in different semen extenders. Semen samples were stored at 17℃ for 96 hours. On everyday (0, 24, 48, 72, 96 h) after storage, the sperm characteristics relevant for fertility, such as sperm motility, viability, acrosome integrity and HOST positive were evaluated. The motility of spermatozoa stored in different extenders was no significantly different among other extenders (P>0.05). Also, no difference was observed among samples processed with different extenders in the percentage of sperm viability, acrosome integrity and HOST positive. All extenders maintained a high percentage (70%) of sperm motility, viability and acrosome integrity through 96 h of storage. The result of this study show that there was no significant differences among extenders in their capacity to preserve motility, viability and membrane integrity of spermatozoa from normal, fertile Korean Native boars for 96 h of liquid preservation at 17℃.

The objective of this study was to investigate the effect of bacterial contamination on elapsed time after preservation on boar semen. Known numbers of Escherichia coli (E. coli) were inoculated to freshly ejaculated semen and sperm parameters such as viability, motility, agglutination, acrosome integrity and hypo-osmotic swelling test were perform-ed during 7 days of liquid preservation. Semen samples were prepared using antibiotic free BTS extender and 4 di-fferent levels of E. coli were treated to semen with following concentrations; 3,000, 5,000, 7,000, 10,000 CFU/ml of sperms. Semen samples were preserved at 17℃ for 7 days in semen storage until analyzed. Aliquots were subjected to measure the sperm viability, motility and agglutination using computer assisted sperm analysis (CASA) system, acrosome integrity was performed using chlortetracycline (CTC) staining method and hypo-osmotic swelling test was performed using hypotonic solution from day 1 (day of semen collection) to 7. Detrimental effects on sperm motility and viability were observed 3 days after preservation at the level of 5,000 CFU/ml (p<0.05). Percentage of sperm abnor-mality was higher (p<0.05) in over 5,000 CFU/ml groups. Sperm agglutination rate was also significantly higher (p< 0.05) in groups of 5,000 and 7,000 CFU/ml. The rate of acrosome reacted sperm was higher as preservation time goes in all the samples but the pattern was clearly higher among E. coli contaminated groups (p<0.05). The sperm mem-brane integrity in terms of hypo-osmotic test, E. coli affects little compared to other sperm parameters. The deleterious effects observed due to the bacterial contamination in semen suggest that importance of hygiene protocol to minimize the bacterial contamination during semen collection and processing.